Comparative analysis of EV isolation procedures for miRNAs detection in serum samples
Zoraida Andreu,Eva Rivas,Aitana Sanguino-Pascual,Amalia Lamana,Mónica Marazuela,Isidoro González-Álvaro,Francisco Sánchez-Madrid,Hortensia de la Fuente,María Yáñez-Mó +8 more
TLDR
Using PEG precipitation in a longitudinal study with human samples, it is demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage and point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses.Abstract:
Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation-based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR-126, miR-30c and miR-143), while the detection of miR-21, miR-16-5p and miR-19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut-off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.read more
Citations
More filters
Journal ArticleDOI
Isolation of Extracellular Vesicles: General Methodologies and Latest Trends.
TL;DR: This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method.
Journal ArticleDOI
Methodological Guidelines to Study Extracellular Vesicles.
Frank A. W. Coumans,Alain Brisson,Edit I. Buzás,Françoise Dignat-George,Esther E.E. Drees,Samir El-Andaloussi,Samir El-Andaloussi,Costanza Emanueli,Costanza Emanueli,Aleksandra Gasecka,Aleksandra Gasecka,An Hendrix,Andrew F. Hill,Romaric Lacroix,Yi Lee,Ton G. van Leeuwen,Nigel Mackman,Imre Mäger,Imre Mäger,John P. Nolan,Edwin van der Pol,D. Michiel Pegtel,Susmita Sahoo,Pia Siljander,Guus Sturk,Olivier De Wever,Rienk Nieuwland +26 more
TL;DR: This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs.
Journal ArticleDOI
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper
Bogdan Mateescu,Emma J. K. Kowal,Bas W.M. van Balkom,Sabine Bartel,Suvendra N. Bhattacharyya,Edit I. Buzás,Amy H. Buck,Paola de Candia,Franklin Wang Ngai Chow,Saumya Das,Tom A. P. Driedonks,Lola Fernández-Messina,Franziska Haderk,Andrew F. Hill,Jennifer Jones,Kendall Van Keuren-Jensen,Charles P. Lai,Cecilia Lässer,Italia Di Liegro,Taral R. Lunavat,Magdalena J. Lorenowicz,Sybren L. N. Maas,Imre Mäger,María Mittelbrunn,Stefan Momma,Kamalika Mukherjee,Muhammad Nawaz,D. Michiel Pegtel,Michael W. Pfaffl,Raymond M. Schiffelers,Hidetoshi Tahara,Clotilde Théry,Juan Pablo Tosar,Marca H. M. Wauben,Kenneth W. Witwer,Esther N. M. Nolte-‘t Hoen +35 more
TL;DR: This position paper was written by the participants of the workshop to give an overview of the current state of knowledge in the field and to clarify that incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research.
Journal ArticleDOI
A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents
Inas Helwa,Jingwen Cai,Michelle Drewry,Arthur D. Zimmerman,Michael B. Dinkins,Mariam Lotfy Khaled,Mutsa Seremwe,W. Michael Dismuke,Erhard Bieberich,W. Daniel Stamer,Mark W. Hamrick,Yutao Liu,Yutao Liu +12 more
TL;DR: Comparing the exosomes extracted by three different exosome isolation kits and differential ultracentrifugation using six different volumes of a non-cancerous human serum shows that three kits are viable alternatives to UC, even when limited amounts of biological samples are available.
Journal ArticleDOI
A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.
Kieran Brennan,Kenneth Martin,Stephen Peter Fitzgerald,James O’Sullivan,Y. Wu,A. Blanco,Ciaran Richardson,Margaret M. Mc Gee +7 more
TL;DR: Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.
References
More filters
Journal ArticleDOI
Extracellular vesicles: exosomes, microvesicles, and friends.
TL;DR: This review focuses on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
Journal ArticleDOI
Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids
TL;DR: This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosomes preparations.
Journal ArticleDOI
Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases
Xi Chen,Yi Ba,Lijia Ma,Lijia Ma,Xing Cai,Yuan Yin,Kehui Wang,Jigang Guo,Yujing Zhang,Jiangning Chen,Xing Guo,Qibin Li,Qibin Li,Xiaoying Li,Wenjing Wang,Yan Zhang,Jin Wang,Xueyuan Jiang,Yang Xiang,Chen Xu,Pingping Zheng,Juanbin Zhang,Ruiqiang Li,Hongjie Zhang,Xiaobin Shang,Ting Gong,Guang Ning,Jun Wang,Jun Wang,Ke Zen,Junfeng Zhang,Chen-Yu Zhang +31 more
TL;DR: It is demonstrated that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses, and can serve as potential biomarkers for the detection of various cancers and other diseases.
Journal ArticleDOI
Biological properties of extracellular vesicles and their physiological functions
María Yáñez-Mó,Pia Siljander,Zoraida Andreu,Apolonija Bedina Zavec,Francesc E. Borràs,Edit I. Buzás,Krisztina Buzas,Krisztina Buzas,Enriqueta Casal,Francesco Cappello,Joana Carvalho,Joana Carvalho,Eva Colas,Anabela Cordeiro da Silva,Anabela Cordeiro da Silva,Stefano Fais,Juan M. Falcón-Pérez,Irene M. Ghobrial,Bernd Giebel,Mario Gimona,Mario Gimona,Michael W. Graner,Ihsan Gursel,Mayda Gursel,Niels H. H. Heegaard,Niels H. H. Heegaard,An Hendrix,Peter Kierulf,Katsutoshi Kokubun,Maja Kosanović,Veronika Kralj-Iglič,Eva-Maria Krämer-Albers,Saara Laitinen,Cecilia Lässer,Thomas Lener,Thomas Lener,Erzsébet Ligeti,Aija Linē,Georg Lipps,Alicia Llorente,Jan Lötvall,Mateja Manček-Keber,Antonio Marcilla,María Mittelbrunn,Irina Nazarenko,Esther N. M. Nolte-‘t Hoen,Tuula A. Nyman,Lorraine O'Driscoll,Mireia Olivan,Carla Oliveira,Carla Oliveira,Éva Pállinger,Hernando A. del Portillo,Hernando A. del Portillo,Jaume Reventós,Jaume Reventós,Marina Rigau,Eva Rohde,Eva Rohde,Marei Sammar,Francisco Sánchez-Madrid,Nuno Santarém,Nuno Santarém,Katharina Schallmoser,Katharina Schallmoser,Marie Stampe Ostenfeld,Willem Stoorvogel,Roman Štukelj,Susanne G. van der Grein,M. Helena Vasconcelos,M. Helena Vasconcelos,Marca H. M. Wauben,Olivier De Wever +72 more
TL;DR: A comprehensive overview of the current understanding of the physiological roles of EVs is provided, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia.
Journal ArticleDOI
Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes
Jean Michel Escola,Monique J. Kleijmeer,Willem Stoorvogel,Janice Griffith,Osamu Yoshie,Hans J. Geuze +5 more
TL;DR: Exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82, and Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs.
Related Papers (5)
Minimal information for studies of extracellular vesicles 2018 (MISEV2018) : a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
Clotilde Théry,Kenneth W. Witwer,Elena Aikawa,María José Alcaraz,Johnathon D. Anderson,Ramaroson Andriantsitohaina,Anna Antoniou,Anna Antoniou,Tanina Arab,Fabienne Archer,Georgia K. Atkin-Smith,D. Craig Ayre,Jean Marie Bach,Daniel Bachurski,Hossein Baharvand,Hossein Baharvand,Leonora Balaj,Shawn Baldacchino,Natalie N. Bauer,Amy A. Baxter,Mary Bebawy,Carla Beckham,Apolonija Bedina Zavec,Abderrahim Benmoussa,Anna Berardi,Paolo Bergese,Ewa Bielska,Cherie Blenkiron,Sylwia Bobis-Wozowicz,Eric Boilard,Wilfrid Boireau,Antonella Bongiovanni,Francesc E. Borràs,Steffi Bosch,Chantal M. Boulanger,Chantal M. Boulanger,Xandra O. Breakefield,Andrew M. Breglio,Andrew M. Breglio,Meadhbh Á. Brennan,Meadhbh Á. Brennan,David R. Brigstock,David R. Brigstock,Alain Brisson,Marike L. D. Broekman,Marike L. D. Broekman,Jacqueline Bromberg,Paulina Bryl-Górecka,Shilpa Buch,Amy H. Buck,Dylan Burger,Sara Busatto,Sara Busatto,Dominik Buschmann,Benedetta Bussolati,Edit I. Buzás,Edit I. Buzás,James Bryan Byrd,Giovanni Camussi,David R. F. Carter,Sarah Caruso,Lawrence W. Chamley,Yu Ting Chang,Amrita Datta Chaudhuri,Chihchen Chen,Shuai Chen,Lesley Cheng,Andrew Chin,Aled Clayton,Stefano P. Clerici,Alex Cocks,Emanuele Cocucci,Robert J. Coffey,Anabela Cordeiro-da-Silva,Yvonne Couch,Frank A. W. Coumans,Beth Coyle,Rossella Crescitelli,Miria Ferreira Criado,Crislyn D'Souza-Schorey,Saumya Das,Paola de Candia,Eliezer F. De Santana,Olivier De Wever,Hernando A. del Portillo,Tanguy Demaret,Sarah Deville,Sarah Deville,Andrew Devitt,Bert Dhondt,Dolores Di Vizio,Lothar C. Dieterich,Vincenza Dolo,Ana Paula Domínguez Rubio,Massimo Dominici,Mauricio Rocha Dourado,Mauricio Rocha Dourado,Tom A. P. Driedonks,Filipe V. Duarte,Heather M. Duncan,Ramon M. Eichenberger,Karin M. Ekström,Samir El Andaloussi,Celine Elie-Caille,Uta Erdbrügger,Juan M. Falcón-Pérez,Farah Fatima,Jason E. Fish,Jason E. Fish,Miguel Flores-Bellver,András Försönits,Annie Frelet-Barrand,Fabia Fricke,Fabia Fricke,Gregor Fuhrmann,Susanne Gabrielsson,Ana Gámez-Valero,Chris Gardiner,Kathrin Gärtner,Raphael Gaudin,Raphael Gaudin,Yong Song Gho,Bernd Giebel,Caroline Gilbert,Mario Gimona,Ilaria Giusti,Deborah C.I. Goberdhan,André Görgens,André Görgens,Sharon M. Gorski,David W. Greening,Julia Christina Gross,Alice Gualerzi,Gopal N. Gupta,Dakota Gustafson,Aase Handberg,Reka A. Haraszti,Paul Harrison,Hargita Hegyesi,An Hendrix,Andrew F. Hill,Fred H. Hochberg,Karl F. Hoffmann,Beth Holder,Harry Holthöfer,Baharak Hosseinkhani,Guoku Hu,Yiyao Huang,Yiyao Huang,Veronica Huber,Stuart Hunt,Ahmed Ibrahim,Tsuneya Ikezu,Jameel M. Inal,Mustafa Isin,Alena Ivanova,Hannah K Jackson,Søren Jacobsen,Steven M. Jay,Muthuvel Jayachandran,Guido Jenster,Lanzhou Jiang,Suzanne M. Johnson,Jennifer Jones,Ambrose Jong,Tijana Jovanovic-Talisman,Stephanie M. Jung,Raghu Kalluri,Shin Ichi Kano,Sukhbir Kaur,Yumi Kawamura,Yumi Kawamura,Evan T. Keller,Delaram Khamari,Elena Khomyakova,Anastasia Khvorova,Peter Kierulf,Kwang Pyo Kim,Thomas Kislinger,Thomas Kislinger,Mikael Klingeborn,David J. Klinke,Miroslaw Kornek,Maja Kosanović,Árpád Kovács,Eva-Maria Krämer-Albers,Susanne Krasemann,Mirja Krause,Igor V. Kurochkin,Gina D. Kusuma,Gina D. Kusuma,Sören Kuypers,Saara Laitinen,Scott M. Langevin,Lucia R. Languino,Joanne Lannigan,Cecilia Lässer,Louise C. Laurent,Grégory Lavieu,Elisa Lázaro-Ibáñez,Soazig Le Lay,Myung-Shin Lee,Yi Xin Fiona Lee,Débora S. Lemos,Metka Lenassi,Aleksandra Leszczynska,Isaac T. S. Li,Ke Liao,Sten F.W.M. Libregts,Erzsébet Ligeti,Rebecca Lim,Rebecca Lim,Sai Kiang Lim,Aija Linē,Karen Linnemannstöns,Alicia Llorente,Catherine Lombard,Magdalena J. Lorenowicz,Ákos M. Lörincz,Jan Lötvall,Jason Lovett,Michelle C. Lowry,Xavier Loyer,Xavier Loyer,Quan Lu,Barbara Lukomska,Taral R. Lunavat,Sybren L. N. Maas,Harmeet Malhi,Antonio Marcilla,Jacopo Mariani,Javier Mariscal,Elena S. Martens-Uzunova,Lorena Martin-Jaular,M. Carmen Martinez,Vilma R. Martins,Mathilde Mathieu,Suresh Mathivanan,Marco Maugeri,Lynda K. McGinnis,Mark J. McVey,David G. Meckes,Katie Meehan,Inge Mertens,Inge Mertens,Valentina R. Minciacchi,Andreas Möller,Malene Jørgensen,Aizea Morales-Kastresana,Jess Morhayim,François Mullier,Maurizio Muraca,Luca Musante,Veronika Mussack,Dillon C. Muth,Kathryn H. Myburgh,Tanbir Najrana,Muhammad Nawaz,Irina Nazarenko,Irina Nazarenko,Peter Nejsum,Christian Neri,Tommaso Neri,Rienk Nieuwland,Leonardo Nimrichter,John P. Nolan,Esther N. M. Nolte-‘t Hoen,Nicole Noren Hooten,Lorraine O'Driscoll,Tina O'Grady,Ana O'Loghlen,Takahiro Ochiya,Martin Olivier,Alberto Ortiz,Luis A. Ortiz,Xabier Osteikoetxea,Ole Ostegaard,Ole Ostegaard,Matias Ostrowski,Jaesung Park,D. Michiel Pegtel,Héctor Peinado,Francesca Perut,Michael W. Pfaffl,Donald G. Phinney,Bartijn C. H. Pieters,Ryan C. Pink,David S. Pisetsky,Elke Pogge von Strandmann,Iva Polakovicova,Ivan K. H. Poon,Bonita H. Powell,Ilaria Prada,Lynn Pulliam,Lynn Pulliam,Peter J. Quesenberry,Annalisa Radeghieri,Robert L. Raffai,Robert L. Raffai,Stefania Raimondo,Janusz Rak,Marcel I. Ramirez,Marcel I. Ramirez,Graça Raposo,Morsi S. Rayyan,Neta Regev-Rudzki,Franz Ricklefs,Paul D. Robbins,David D. Roberts,Silvia C Rodrigues,Eva Rohde,Sophie Rome,Kasper M.A. Rouschop,Aurelia Rughetti,Ashley E. Russell,Paula Saa,Susmita Sahoo,Edison Salas-Huenuleo,Catherine Sánchez,Julie A. Saugstad,Meike J. Saul,Raymond M. Schiffelers,Raphael Schneider,Tine Hiorth Schøyen,Aaron Scott,Eriomina Shahaj,Shivani Sharma,Olga Shatnyeva,Faezeh Shekari,Ganesh Vilas Shelke,Ashok K. Shetty,Kiyotaka Shiba,Pia Siljander,Andreia M. Silva,Agata Skowronek,Orman L. Snyder,Rodrigo Pedro Soares,Barbara W Sódar,Carolina Soekmadji,Carolina Soekmadji,Javier Sotillo,Philip D. Stahl,Willem Stoorvogel,Shannon L. Stott,Erwin Strasser,Simon Swift,Hidetoshi Tahara,Muneesh Tewari,Kate Timms,Swasti Tiwari,Swasti Tiwari,Rochelle Tixeira,Mercedes Tkach,Wei Seong Toh,Richard Tomasini,Ana Claudia Torrecilhas,Juan Pablo Tosar,Juan Pablo Tosar,Vasilis Toxavidis,Lorena Urbanelli,Pieter Vader,Bas W.M. van Balkom,Susanne G. van der Grein,Jan Van Deun,Martijn J. C. van Herwijnen,Kendall Van Keuren-Jensen,Guillaume van Niel,Martin E. van Royen,Andre J. van Wijnen,M. Helena Vasconcelos,Ivan J. Vechetti,Tiago Degani Veit,Laura J Vella,Laura J Vella,Emilie Velot,Frederik J. Verweij,Beate Vestad,Jose L. Viñas,Tamás Visnovitz,Krisztina V. Vukman,Jessica Wahlgren,Dionysios C. Watson,Marca H. M. Wauben,Alissa M. Weaver,Jason P. Webber,Viktoria Weber,Ann M. Wehman,Daniel J. Weiss,Joshua A. Welsh,Sebastian Wendt,Åsa M. Wheelock,Zoltán Wiener,Leonie Witte,Joy Wolfram,Joy Wolfram,Joy Wolfram,Angeliki Xagorari,Patricia Xander,Jing Xu,Xiaomei Yan,María Yáñez-Mó,María Yáñez-Mó,Hang Yin,Yuana Yuana,Valentina Zappulli,Jana Zarubova,Jana Zarubova,Vytautas Žėkas,Jian ye Zhang,Zezhou Zhao,Lei Zheng,Alexander R. Zheutlin,Antje M. Zickler,Pascale Zimmermann,Pascale Zimmermann,Angela M. Zivkovic,Davide Zocco,Ewa K. Zuba-Surma +417 more
Biological properties of extracellular vesicles and their physiological functions
María Yáñez-Mó,Pia Siljander,Zoraida Andreu,Apolonija Bedina Zavec,Francesc E. Borràs,Edit I. Buzás,Krisztina Buzas,Krisztina Buzas,Enriqueta Casal,Francesco Cappello,Joana Carvalho,Joana Carvalho,Eva Colas,Anabela Cordeiro da Silva,Anabela Cordeiro da Silva,Stefano Fais,Juan M. Falcón-Pérez,Irene M. Ghobrial,Bernd Giebel,Mario Gimona,Mario Gimona,Michael W. Graner,Ihsan Gursel,Mayda Gursel,Niels H. H. Heegaard,Niels H. H. Heegaard,An Hendrix,Peter Kierulf,Katsutoshi Kokubun,Maja Kosanović,Veronika Kralj-Iglič,Eva-Maria Krämer-Albers,Saara Laitinen,Cecilia Lässer,Thomas Lener,Thomas Lener,Erzsébet Ligeti,Aija Linē,Georg Lipps,Alicia Llorente,Jan Lötvall,Mateja Manček-Keber,Antonio Marcilla,María Mittelbrunn,Irina Nazarenko,Esther N. M. Nolte-‘t Hoen,Tuula A. Nyman,Lorraine O'Driscoll,Mireia Olivan,Carla Oliveira,Carla Oliveira,Éva Pállinger,Hernando A. del Portillo,Hernando A. del Portillo,Jaume Reventós,Jaume Reventós,Marina Rigau,Eva Rohde,Eva Rohde,Marei Sammar,Francisco Sánchez-Madrid,Nuno Santarém,Nuno Santarém,Katharina Schallmoser,Katharina Schallmoser,Marie Stampe Ostenfeld,Willem Stoorvogel,Roman Štukelj,Susanne G. van der Grein,M. Helena Vasconcelos,M. Helena Vasconcelos,Marca H. M. Wauben,Olivier De Wever +72 more